1Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel.

Abstract

Understanding the molecular triggers of pancreatic β-cell proliferation may facilitate the development of regenerative therapies for diabetes. Genetic studies have demonstrated an important role for cyclin D2 in β-cell proliferation and mass homeostasis, but its specific function in β-cell division and mechanism of regulation remain unclear. Here, we report that cyclin D2 is present at high levels in the nucleus of quiescent β-cells in vivo. The major regulator of cyclin D2 expression is glucose, acting via glycolysis and calcium channels in the β-cell to control cyclin D2 mRNA levels. Furthermore, cyclin D2 mRNA is down-regulated during S-G(2)-M phases of each β-cell division, via a mechanism that is also affected by glucose metabolism. Thus, glucose metabolism maintains high levels of nuclear cyclin D2 in quiescent β-cells and modulates the down-regulation of cyclin D2 in replicating β-cells. These data challenge the standard model for regulation of cyclin D2 during the cell division cycle and suggest cyclin D2 as a molecular link between glucose levels and β-cell replication.

Cyclin D2 is present in the nucleus of most β-cells in vivo. A, Immunostaining for cyclin D2 in the pancreas of 1-wk-old wild-type (WT) (top) and cyclin D2−/− (KO) (bottom) mice. The absence of signal in mutant pancreas indicates that nuclear staining in wild-type animals reflects true cyclin D2 protein. B, Immunostaining for cyclin D2 in acinar cells of 1-wk-old wild-type (top) and cyclin D2−/− (bottom) mice. As with islets, in the acinar, there is no cyclin D2 signal in the knockout mouse. C, Costaining for cyclin D2 (red), the proliferation marker PCNA (blue), and insulin (green) in 1-month-old mice shows that cyclin D2 is present in the nucleus of most β-cells, even though only a small fraction of the cells is proliferating. D, Costaining for cyclin A (red), BrdU (blue), and insulin (green) in 1-month-old mice injected with BrdU 2 h before killing, showing that only proliferating cells express cyclin A.

Cyclin D2 is down-regulated in replicating β-cells via a glucose-sensitive mechanism. A, Costaining for cyclin D2 and BrdU reveals that replicating β-cells, as well as replicating acinar cells, do not express cyclin D2. Image is taken from a 1-month-old mouse, injected with BrdU 2 h before killing. B, Quantification of the fraction of cyclin D2+ β-cells in BrdU− β-cells in vivo and in BrdU+ β-cells in mice injected with BrdU 2 or 7 h before killing. At least three animals with a minimum of 1500 β-cells (∼75 Brdu+ cells) were analyzed. ANOVA yielded P < 0.01. C, Enhanced glucose metabolism and intracellular calcium prevents the decrease in cyclin D2 in replicating β-cells. One-month-old mice were injected with BrdU 8 h before killing. Seven hours before killing, mice were injected with a small molecule GKA and the calcium channel opener BayK8644. At least three animals with a minimum of 1500 β-cells (∼75 Brdu+ cells) were analyzed. ANOVA yielded P < 0.01. D, Islet cells during S-G2-M phases of the cell cycle (GFP+) show reduced levels of cyclin D2 and GCK mRNA.